C-type natriuretic Peptide (CNP), the third natriuretic peptide (NP) identified, is mainly expressed in the nervous system and endothelial cells. In addition, CNP is believed to be produced locally in tubular cells and glomeruli of normal human kidneys. CNP exerts mainly vasodilatory and anti-mitogenetic effects rather than regulation of body fluid homeostasis via autocrine or pa­racrine pathway. Many factors, such as shear, pro-inflammatory cytokines and lipopolysaccharide, can regulate the production and excretion of CNP both in vivo and in vitro. However, little infor­mation about the renal action of CNP was obtained in the past from the model of isolated perfused rat kidney in which variables could be changed in a controlled manner and systemic influences could be eliminated. However, reviewing the data from the studies that used this model inspires us to conclude that such model can be a useful tool to probe the undiscovered aspects of the renal actions of CNP and should be advocated for future studies on it.

The Natriuretic Peptide (NP) system is a family of hormones with similar molecular structure and physiological function involved in regula­tion of blood pressure, electrolyte, and volume homeostasis.[1] Moreover, NP system also plays a significant role in inhibiting vascular remo­deling after injury. [2] The study of NP began 50 years ago, when electron microscopy revealed the presence of electron-dense "specific atrial granules" in cardiac atrial cells containing at­rial natriuretic peptide (ANP) as confirmed by de Bold in the early 80s.[3] From 1998 to 1990, two more members of the NP family, the brain natriuretic peptide (BNP) and C-type natriure­tic peptide (CNP) were identified in porcine brain.[4],[5] Other structurally related peptides in­clude Dendroaspis Natriuretic Peptide (DNP) from the venom of the green mamba, Dendro­aspis angusticeps ,[6] urodilatin, which is a pro­duct of alternative processing of pro-ANP by renal cell,[7] and guanylin and uroguanylin, which were isolated from rat intestine and opossumurine, respectively.[8],[9]

Two CNP fragments with 22 and 53 amino acids are derived from a 103 amino acid CNP precursor; the former sequence being contained within the latter. The 22 amino acid fragment may be regarded as the mature and more biologically active form [Figure 1].

The major sites of CNP expression are the nervous system and endothelial cells.[1],[10] In ad­dition, CNP is believed to be produced locally in tubular cells and glomeruli of normal human kidneys.[11] Unlike the closely related hormone ANF and BNF, CNF does not enter the circula­tion in significant amounts, and it is regarded as an autocrine or paracrine factor in the tissue and organs in which it is expressed, such as kidneys.[12] CNP elicits mainly vasodilatory ef­fects rather than regulation of body fluid ho­meostasis. The different physiological effects of these peptides could be attributed to the existence of three different receptors subtypes, natriuretic peptide receptors A, B, and C (NPR­A, NPR-B, NPR-C).[13] NPR-A is a guanylate cyclase-coupled receptor activated by ANP, BNP and DNP. NPR-B is also a guanylate cy­clase-coupled receptor, which specifically binds CNP and results subsequently in an elevation in intracellular cyclic guanosine monophos­phate (cGMP). NPR-C is a truncated protein with an extracellular natriuretic binding domain but only a short 37 amino acid intracellular tail that possesses a G-protein activating sequence. Two different subtypes of NPR-C, of approxi­mately 67 and 77 KD in size have been iden­tified. In general, the main function of the 77 KD NPR-C appears to act as a clearance path­way by binding the NPs and internalizing them, whereas the 67 KD NPR-C is probably in­ volved in adenylyl cyclase inhibition.[14],[15] These roles are supported by the hypertensive status of NPR-C knockout mice as a result of in­creased serum concentrations of ANP, BNP and cyclic adenosine monomphsopate (cAMP).[16] The NP system also is inactivated by neutral endopeptidases present within renal tubular cells and vascular cells [Figure 2].

Numerous studies have probed the renal ac­tion of CNP in vivo and in vitro. CNP is a po­tent relaxant of vascular smooth muscle and anti-mitogenetic, but has little effect on salt and water excretion.[17] Lee et al [18] reported that the administration of exogenous CNP (29.3 ng/ kg/min Χ 75min) to seven healthy dogs via intravenous drip could significantly increase serum cGMP [7.8 ± 0.8 pmol/mL (pre-infusion) to 10.1 ± 0.6 pmol/mL (at 60 min of infusion)] and urine flow [0.5 ± 0.1 mL/min (pre-infu­sion) to 1.3 ± 0.3 mL/min (at 60 min of infu­sion)], whereas no obvious changes of urinary sodium excretion and glomerular filtration rate (GFR) were observed; these results were con­sistent with another study in humans.[19] CNP produces vasodilatation of the afferent arte­riole via the release of cGMP and nitric oxide (endothelial-derived relaxing factor, EDRF), and vasoconstriction of efferent arteriole selectively, which may attribute to the different distribu­tion of NP receptors in kidney.[20],[21] Furthermore, CNP exerts inhibitory effects on rat mesangial cell proliferation both in vivo and in vitro.[22],[23] Many factors, such as shear, pro-inflammatory cytokines and lipopolysaccharide, can regulate the renal action of CNP.[24],[25],[26] Woodward et al [25] have shown that the production and secretion of CNP in vascular smooth muscle cells (VSMC) was stimulated by transforming growth factor-β(TGF-β), whereas basic fibroblast growth factor (bFGF) played an inhibitory role. In a more recent study, Mendonca et al [27] found that platelet-derived growth factor-BB (PDGF-BB) also induced significant dose-dependent increa­ses in CNP transcript of VSMC in vitro.

Nevertheless, little information about the renal action of CNP is obtained from the model of isolated perfused rat kidney, in which variables can be changed in a controlled manner and systemic influences can be eliminated. There­fore, the use of this model to probe the undis­covered aspects of CNP seems to be appealing.

Advantages of the Isolated Perfused Rat Kidney Model

The present perfusion techniques for studies of whole organs are derived from those deve­loped in 1950s by Weiss et al.[28] In the past five decades, the isolated perfused rat kidney was considered as a suitable model for the study of many physiological and biochemical aspects of renal function.[29] The advantages of the mo­del are obvious. First, it allows for the direct study of the pre and postglomerular microcir­culation with minimal trauma to the vessels or parenchymal tissues. Second, neural and hor­monal influences on the renal circulation are eliminated, and perfusion pressure and compo­sition can be controlled. Third, vasoactive agents can be administered to the perfusate or bath. Fourth, tubular and vascular relationships are preserved, and tubuloglomerular feedback sys­tem is intact. Fifth, the network basis of the circulation is largely preserved. Finally, pre­ssures, flows, and vascular diameters can be directly measured.[30],[31]

Data from the Studies on the NP System in Isolated Perfused Rat Kidney

Burton et al [32] measured cGMP release and vascular tone in the isolated kidney of the rat perfused at constant flow with Krebs-Henseleit solution More Details. The effect of ANP (0.01΅M) on the renal release of cGMP and vascular tone was examined. They observed that CNP signifi­cantly inhibited the vasoconstrictor response to methoxamine, and produced an immediate and sustained effect on cGMP release, which in­creased from 0.63 ± 0.36 to 5.80 ± 1.96 pmol/ min during the four to six min period of the ANP infusion. However, both the increase in cGMP release produced by ANF in the isolated perfused kidney and its renal vasodilator acti­vity remained unaffected by treatment with he­moglobin, which acts as an EDRF inhibitor by binding nitric oxide.[33] EDRF synthesis was not involved in either the renal vasodilator activity of ANF or its ability to stimulate cGMP syn­thesis, which was contrary to the report of Green et al[34] in cultured human glomerular endothe­lial cells.

Heringlake et al[35] investigated the relationship between arterial blood pressure and renal uro­dilatin excretion in an isolated perfused rat kidney model. An increase of urodilatin excre­tion was observed if renal perfusion pressure was raised from 80 to 120 mmHg, accompa­nied by increased urine flow, urinary sodium, and urinary potassium, whereas the concentra­tion of urodilatin in the perfusate did not change significantly, and serial measurements revealed no direct relation of urodilatin excretion with either urine flow or urinary sodium. This sug­gests that renal perfusion pressure is a deter­minant of urodilatin excretion, and urinary and venous effluent concentrations of urodilatin (pro­bably production) are not coupled directly. Fur­thermore, urodilatin excretion and urinary so­dium may dissociate during acute variations of sodium excretion and urine flow in contrast to the in vivo findings of Schmidt et al.[36]

In another study, Fonteles et al[37] compared the biological effects of guanylin and urogua­nylin in the isolated perfused rat kidney. They found that both guanylin and uroguanylin in­creased GER, osmolar clearance, kaliuresis and urinary sodium, with the reduction in fractio­nal sodium reabsorption, but guanylin appeared to be less potent than uroguanylin. Moreover, there was also an experiment designed in the isolated perfused rat kidney to identify possi­ble synergisms between ANP, guanylin and uro­guanylin.[38] These experimental results showed that guanylin promoted a reduction in fractio­nal sodium reabsorption, after the pretreatment with ANP. In contrast, ANP blocked urogua­nylin-induced increase in urine flow and the reduction in fractional sodium reabsorption. Thus, the synergism between ANP + guanylin and the antagonism between ANP + urogua­nylin may be attributed to the existence of dif­ferent subtypes of receptors mediating the renal actions of guanylins.

After reviewing the data above, we can draw a conclusion that the isolated perfused rat kid­ney is one of the useful tools to investigate the undiscovered aspects of NP system including physiological effects, pathophysiological indi­cators, potential therapeutic actions, and inter­actions with other hormones in the kidney.

Applied Prospects for the Studies on the Renal Action of CNP in Isolated Perfused Rat Kidney

CNP does not circulate in the blood in appre­ciable amounts and therefore may act locally in an autocrine or paracrine way. Although little information about the renal action of CNP is obtained from the model of isolated perfused rat kidney, we can presume its applied pros­pects, depending on the availability of the data from the studies on other NP members in iso­lated perfused rat kidneys and from the fol­lowing studies on CNP both in vivo and in vitro.

Involvement of CNP in the Regulation of Water­Electrolyte Homeostasis

In anesthetized rats,[39] conscious sheep,[40] and normal humans,[19] CNP has been shown to be mildly diuretic and natriuretic. However, CNP paradoxically acts as an antinatriuretic subs­tance. This has been suggested by Stingo and colleagues[41] who observed a significant dec­rease in natriuresis with systemic CNP infu­sion in the anesthetized dog. The different in­volvement of CNP in the regulation of water­electrolyte homeostasis may attribute to the diversities of the experimental conditions in the different species.

Effect of CNP on Renal Hemodynamics

CNP is widely present in endothelium and plays a role in the regulation of vascular tone. Garcha et al[42] demonstrated that CNP relaxes human isolated subcutaneous resistance arte­ries by local activation of cGMP. On the con­trary, when infused in human in vivo, CNP caused no changes on GFR, renal plasma flow (FPR), and renal vascular resistance other than the increased excretion of urinary cGMP.[19] Therefore, the model of isolated perfused rat kidney should be warranted to elucidate the effect of CNP on renal hemodynamics.

Responsibility of CNP to Pressure

A recent article in circulation [43]reports that CNP levels are elevated in the hearts of pa­tients with congestive heart failure, which in­dicates that locally synthesized CNP in the heart may then compensate for the hyperten­sion induced by fluid volume retention. This date is also supported by the results of Gulberg et al, [44] which showed an increase in urinary CNP excretion in patients with cirrhosis and functional renal failure. To further investigate the origin of increased urinary CNP excretion, CNP plasma concentrations from a systemic artery and renal vein were simultaneously mea­sured in this study. Almost identical concen­trations in these two vessels exclude major renal extraction of circulating CNP of systemic origin, which suggests enhanced renal CNP production in cirrhotic patients with hepatore­nal syndrome. In addition, more direct evidence from the report of Kim et al [45] indicates that the expression of CNP mRNA was increased in the kidney of obstructive uropathic rats. Diffe­rent renal perfusion pressure should be held to test the responsibility of CNP in isolated per­fused rat kidney.

Influence of CNP on Glomerular Permeability

Nephrotic Syndrome (NS) is characterized by alterations of permselectivity at the glomerular capillary wall, resulting in its inability to res­trict the urinary loss of protein.[46] The study of Cataliotti et al [47] illustrated a positive correla­tion between urinary CNP and albuminuria in patients with NS, and the percent reduction of urinary CNP after low-protein diet was signi­ficantly greater than the percent reduction in albuminuria. Therefore, renal release of CNP may alter the glomerular permeability and may be sensitive to manipulations in glomerular function occurring in clinical practice.

Cross-Talking Between CNP and Other NP Members

An inverse relationship between circulating CNP and ANP was observed by Gulberg et al [44] in normal humans. However, intrinsic effects or cross-talking of these peptides, including regulation of ANP release via a short feedback loop, have not yet been clearly defined until a study on perfused atria was conducted by Lee et al [48] who postulated that CNP suppresses the secretion of ANP via the particular NPR-B­cGMP pathway. Therefore, a similar study on isolated perfused rat kidney should be under­taken to clarify the cross-talking between CNP and other NP members.

Antagonism between CNP and the Renin­Angiotensin-Aldosterone System (RA-AS)

CNP exerts a renoprotective role via inhibi­tion of the RAAS in pathophysiological situa­tions, and has been shown to offer a therapeu­tic advantage in the treatment of kidney di­seases.[15] A recent study has reported that expre­ssions of rennin, angiotensin converting enzyme 1 (ACE1), and CNP mRNA were significantly increased in the rat glomerulus by bilateral ure­teral obstruction, which was hypothesized to have compensatory effects offsetting the hyper­tension induced by fluid retention.[49]

Interaction of CNP with Cytokines

The expression of CNP in vitro is influenced by several cytokines. Dio et al [50] have revealed that the endothelial secretion of CNP is stimu­lated by TGF-β and tumor necrosis factor-a, which not only causes growth inhibition of VSMC at the G1 phase but also induces the re­differentiation of VSMC and the acceleration of endothelial regeneration. Moreover, a num­ber of studies also demonstrated that CNP ex­pression is enhanced by other cytokines, in­cluding interleukin-1, -6, PDGF, and lipopoly­ saccharide.[23],[27],[51] Conversely, CNP expression is inhibited by oxidized low density lipopro­tein, vascular endothelial growth factor, and bFGF.[25],[52],[53] These results should be confirmed by the studies on isolated perfused rat kidney.

Conclusions

Little information about the renal action of CNP is obtained from the model of isolated perfused rat kidney. After comprehending the advantages of the isolated perfused rat kidney model, reviewing the data from the studies on other NP members in isolated perfused rat kidney, and hypothesizing the applied prospects for the studies on the renal action of CNP in isolated perfused rat kidney, we can conclude that the isolated perfused rat kidney is one of the useful tools to investigate the renal action of CNP and should be advocated for future research on it.

Acknowledgement

The authors would like to gratefully acknow­ledge the most helpful comments on this review received from Professor Shi Jun Zhang, De­partment of physiology, Gunagxi Medical Uni­versity, Nanning.

Schweitz H, Vigne P, Moinier D, Frelin C, Lazdunski M. A new member of the natriuretic peptide family is present in the venom of the green mamba (Dendroaspis angusticeps). J Biol Chem 1992;267:13928-32.